PLANT TISSUE CULTURE
DR.NISHI MATHUR ITS METHOD AND
APPLICATIONS
WHAT IS TISSUE CULTURE?
TISSUE CULTURE is the term used for “the process of
growing cells artificially in the laboratory.
PLANT TISSUE CULTURE- Technique of growing plant cells, tissues, and organs in an artificially prepared
nutrient medium under aseptic condition.
Plant cells are totipotent.
Origin : Early 20th century with the work of Gottleib Haberlandt (plants).
REQUIREMENT OF TISSUE CULTURE:
Appropriate tissue (explant)
• A suitable growth medium (Some of the common media are MS, LS,
Gamborg B5, White’s, Hellar for haploids etc)
• Growth regulators
• Aseptic (sterile) conditions
• Frequent subculturing :
TYPES OF PLANT TISSUE CULTURE
• SEED CULTURE
• EMBRYO CULTURE
• MERISTEM CULTURE
• BUD CULTUR
• CALLUS CULTURE
• CELL SUSPENSION CULTURE
• ANTHER CULTURE/OVARY CULTURE
• PROTOPLAST CULTURE
PROCESS OF PLANT TISSUE CULTURE
Selection of explant from mother plant
Parent plant Inoculation Light &warmth
for shoot initiation
Shoots are transferred to rootingmedia(hig her auxin conc)
Shoot
formation Callus induction Plants are kept
for hardening in green house
,sold to a nursery and then pottedup.
Tissue culture plants sold to a nursery & then potted up
The rooted shoots are potted up (deflasked) and
‘hardened off’
This is necessary as many young tissue culture plants have no waxy cuticle to prevent water loss.
FACTORS AFFECTING TISSUE CULTURE
• Growth Media
–Minerals, Growth factors, Carbon source, Hormones
• Environmental Factors
–Light, Temperature, Photoperiod, Sterility, Media
• Explant Source
–Usually, the younger, less
differentiated explant, the better for tissue culture
–Different species show differences in amenability to tissue culture
TISSUE CULTURE APPLICATIONS
Micropropagation
Germplasm preservation
Somaclonal variation
Haploid & dihaploid production
In vitro hybridization – protoplast fusion
Embryo rescue
Synthetic seed production
MICRO-PROPAGATION
Large scale production of genetically identical clones in vitro is called Micro-propagation.
ADVANTAGES:
•Millions of shoot tips from a small piece of plant tissue in a short period of time and space.
•Maintain Genetically uniform progeny.
•Can produce exact copies of plants required that have desirable traits.
•Supplies plant throughout the year irrespective of seasonal variation
COMMERCIAL APPLICATION OF MICROPROPAGATION
• Micro propagation of Orchids demonstrated profit all over the world.
• Many other crops like banana, papaya,
potato, gerbera, carnation, Anthurium, apple, pear,cherry etc have been grown through
micro propagation are commercially exploited.
• Development of disease resistant crops .Some valuable clones recovered from virus are
• Tobacco- TMV, Sugarcane-Mosaic virus etc.
• Among the innovative techniques of micro propagation, the concept of
somatic embryogenesis with synthetic seed production or artificial seed
technology is very promising
GERMPLASM CONSERVATION
• Maintenance of germplasm of plants vulnerable to pathogen, insects and various climatic hazards and plants with early embryo degeneration, are difficult to maintain.
• Plant species which are endangered, rare threatened with extinction are needed to be conserved by
ex-citu method of germplasm conservation.
• Provides cost effective alternative to growing plants under field condition , nurseries or green houses.
• Cryopreservation of germplasm is really effective in conservation biotechnology.
HAPLOID&DIHAPLOID PRODUCTION
• Anther or Ovary/ovule culture
• Production of pure homozygous line in leser time period.
• Androgenic haploids are used to produce different lines of aneuploids like
monosomic,nullisomic,trisomic etc.
• Induction of mutagenesis
• Recessive traits(e.g low glucosinolate in Brassica ) can be made expressed.
• Use in hybrid development and early release of varieties.
ARTIFICIAL SEED PRODUCTION
.
Synthetic or Artificial Seeds (living seed-like structure) are any totipotent cells(somatic embryos or meristem tips etc )which is artificially encapsulated by chemicals (Hydrogels) which behave like true seeds if grown in soil and can be used as substitutes of true seeds and possess
the ability to convert into a plant ex vitro or invitro.
SOMATIC EMBRYO
ARTIFICIAL SEED COAT ARTIFICIAL
ENDOSPERM
GERMINATED SYNTHETIC SEEDS
•Using embryo culture seed dormancy can be overcome.
•Embryo rescue which can circumvent post zygotic barriers.
•To raise the rare hybrids by rescuing embryo from incompatible crosses is the most popular application. The
production of embryos from somatic or
“non-germ” cells.
•Usually involves a callus intermediate stage which can result in variation
among seedlings
EMBRYO CULTURE
PROTOPLAST FUSION
• Production of somatic hybrids.
• Production of CMS line.
• Somatic Hybrids with specific alien gene.
• Production of Unique Nuclear Cytoplasmic Combination.
• Production of Autotetraploid or Allotetraploid.
• Production of Heterozygous line.
•Play an important role in polyploidisation.
•Genetic variability can be raised which include resistance to a particular disease, herbicide
resistance, stress tolerance etc
SOMACLONAL VARIATION
ADVANTAGES OF PLANT TISSUE CULTURE I
• fwith higher productivity if its genome is changed.Parsetvceonmtiomneorcf isatlaprrvoaptiaognatinionthoirfdnewworcldultciovuanrst.ries
• Tmaoktihnegrapnlaenxtp,lasontradroeesanndoteunsduaanllgyerdeedstprolayntths ecan be
cloned safely.
• Oanccoenteisntuaobulisshseudp,palypolfanytoutnisgsupelacnutltsutrherloinueghcoaunt gtihvee year.
• Tdesirable traits.pahretipcruoladrulcytigoonodofflcolowneerss,ofrpuliatsn,tosrthhaatveprootdhuerce
ADVANTAGES OF PLANT TISSUE CULTURE II
• The regeneration of whole plants from plant cells that have been genetically modified.
• Tissue culture clones are ‘true to type’ as
compared with seedlings, which show greater variability.
• The production of plants in sterile containers reduces disease transmission. To clean
particular plants of viral and other infections and to quickly multiply these plants as
'cleaned stock' for horticulture and agriculture.
ADVANTAGES OF PLANT TISSUE CULTURE III
• The production of multiples of plants in the absence of seeds or
necessary pollinators to produce seeds.
• Allows production of plants from seeds that otherwise have very low chances of germinating and growing,
i.e.: Orchids and Nepenthes.
DISADVANTAGES OF PLANT TISSUE CULTURE
• Chromosomal abnormalities appear as cultures age:
undesirable changes.
• Expensive,labour intensive especially if less than 1000 plants are needed.
• The procedure is quite variable and also it depends on the type of the species so sometimes it needs
trial-and-error type of experiments if there is no available review about that species.
Continued..
The procedure needs special attention and diligently done observation.
There may be error in the identity of the organisms after culture.
Infection may continue through generations easily if possible precautions are not taken
Decrease genetic variability.
ADVANTAGES OF TISSUE CULTURE IN CROP
IMPROVEMENT I
• Tissue culture has been exploited to create
genetic variability from which crop plants can be improved.
• to improve the state of health of the planted material and to increase the number of
desirable germplasms available to the plant breeder
• The culture of single cells and meristems can be effectively used to eradicate pathogens
from planting material and thereby
dramatically improve the yield of established cultivars.
ADVANTAGES OF TISSUE CULTURE IN CROP
IMPROVEMENT II
• Large-scale micropropagation laboratories are providing millions of plants for the
commercial ornamental market and the agricultural, clonally-propagated crop
market. With selected laboratory material typically taking one or two decades to
reach the commercial market through plant breeding, this technology can be
expected to have an ever increasing impact on crop improvement as we approach the new millenium.
Research on some horticulture crops
1.POTATO
Solanum tuberosum
Tissue culture plants are when grown In insect proof net houses
produces mini tubers.
Tissue culture of mini tubers give 20-30%
higher yield.
Protected against bacterial disease aphids(bacterial
disease-crop failure).
Uniform evenly shaped potatoes.
• Disease & pest free planting material.
• Little space needed for
multiplying large number of plants
• Plantlets more vigourous ,
uniform shorter harvest period, higher bunch weights.
PLUMUl SHOOT
E TIP
EMBRYO
GERMINATION WELL DEVELOPED ROOT & SHOOT
COMPLETE PLANTLET
ACCLIMITI ZA-TION STATE
SECONDA RY
HARDENIN G
• Allows for faster distribution of superior
germplasm.
EMBRYO CULTURE OF WILD
EXPLANT-SUCKER
5CM PLUG PLANTS ARE GROWN IN GREEN HOUSE
BANANA
FRUITS
PINEAPPLE
• Resistance to mealy
bugs,nematodes,wilt disease,heart and root rot.
• Uniform yield,uniform fruit weighing.
• Faster growth and spineless darker leaves
• Sweeter pulp
• Ripens evenly and naturally
PAPAYA
Rapid and mass propagation of homogenous and uniform plants for both commercial and research purposes.
• As the sex of the plant is known,the required
nummber of male to female plant species can be produced.
• To decrease mortality% and to increase gaermination%.
• To increase yield.
• To avoid papaya viral disease.
• To obtain export quality food.
FLOWERS
SUNFLOWER
• IMMATURE EMBRYO CULTURE- For the shortening of the entire lifecylce
• To overcome seed dormancy.
• In vitro tissue culture methods in sunflower accelerates the
breeding process.
• Gene from sunflower known as HAHB4, which helps the plant endure water shortages. That gene is introduced into wheat, soybean and maize.
Gerbera – hybrid seed germination followed by axillary bud proliferation and separation
GERBERA
ORCHIDS
• Seeds are exceedingly small with small
reserves,loose their viability very fast so they need to be germinated in a nutrient medium.
• Higher seed germination % even in rare to germinate species
• Exact clones are produced .
• Mass and rapid propagation.
• Diseased free plant
• Embryo rescue and culture in hybrid plant production.
ORCHIDS
CAPSULE OF ORCHID
MATURE CAPSULE BEARING SEEDS
CALLUS (OR PLB)OF ORCHID
INVITRO GERMINATION BY SEEDS
Callus culture of Orchids
(a) Clump formation at the base of leaf segments on week three, (b) PLB initiation, (c) PLB development into shoots and roots, (d) direct shoot
formation (e) shoot induced roots on week six and (f) 3-month-old plantlets
Conclusion
• Plant tissue culture represents most promising areas of application and giving an outlook into the future.
• While tissue-culture offers tremendous benefits, it could be a bane if undertaken improperly.
• It could be the fastest means of spreading disease in any crop. If the source of the material to be tissue-
cultured is disease-infected, the resulting tissue-cultured plantlets will carry the disease.
• Since tissue culture could produce thousands if not
millions of plantlets tremendously fast, the disease could spread super fast. Thus conducting tissue culture we
must be sure that the parent plant is free from infection or disease.
THANK YOU
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